Methods are known to provide recombined combinatorial gene expression libraries by crossing and recombination between cells comprising expression constructs (WO 00/52180 Terragen Discovery Ltd). Through the recombination, which may be carried out in vitro using the recA recombination enzyme, novel genes are obtained, which may or may not be functional in the host cell.
One drawback of the libraries of the prior art is that evolution of the libraries may only be obtained through crossing and recombination between cells whereby homologous or homeologous genes are recombined thereby resulting in novel genes yielding gene products with slightly changed properties such as substrate specificity, solubility, cellular location etc.
Furthermore once the expression constructs have been inserted into the cells the specific gene combinations of a cell is static. Novel combinations may be obtained by crossing and recombination, but this will also lead to formation of novel genes through cross-over. The novel genes may or may not be functional anymore.
Furthermore, the expression of the inserted expression construct is a co-expression of all the genes inserted into any one cell. When a large number of heterologous genes from a wide variety of distantly related species is assembled in one cell, chances are great that some of the heterologous genes are lethal or sub-lethal to the cell, or that several gene products will compete for the same substrates. When only co-expression of the inserts is possible novel metabolic pathways may remain undiscovered due to this fact or due to the fact that the novel metabolite was being further metabolised to a known metabolite by another inserted enzyme.